Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/806—Antigenic peptides or proteins
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/808—Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/82—Proteins from microorganisms
  • Y10S530/826—Viruses

Definitions

• the present invention relates to an improved process for the isolation and purification of the hepatitis B surface antigen (HBsAg) for vaccine purposes and the preparation of vaccines derived therefrom.
• HBsAg antigen obtained by the process of the invention is in the form of HBsAg particles measuring from 17 to 20 nm. It is of interest for the preparation of vaccines.
• Hepatitis B is a worldwide and potentially fatal viral disease. Although its etiological agent - the hepatitis B virus - has long been isolated from the blood of patients with the disease, the development of a commercial hepatitis B vaccine has encountered major difficulties: the hepatitis B virus is in the form of a 42 nm particle called Dane particle comprising (a) a nucleus containing the viral genome linked to the nucleocapsid protein and containing the nucleocapsid antigen (HBcAg) which is not immunogenic and (b) an envelope containing the immunogenic surface antigen (HBsAg).
• Dane particle comprising (a) a nucleus containing the viral genome linked to the nucleocapsid protein and containing the nucleocapsid antigen (HBcAg) which is not immunogenic and (b) an envelope containing the immunogenic surface antigen (HBsAg).
• HBsAg is made up of a group of morphologically heterogeneous complex macromolecular structures; it contains proteins, carbohydrates, glycoproteins and lipids, the main constituents being phosphatidylcholine, cholesteryl ester, cholesterol and triglycerides.
• Infection with the hepatitis B virus (HBV) results not only in the production of Dane particles but also in an overproduction of 22 nm particles and filaments containing the elements of the surface envelope. It is known that these 22 nm particles are approximately 1000 times more immunogenic than the monomeric HBsAg protein, but the reproduction and expression of the hepatitis B virus (HBV) has been hampered for a long time by the absence of cell culture systems.
• HBsAg HBsAg type molecules
• HBsAg HBsAg in other host systems
• recombinant DNA technique the expression of HBsAg has been reported from yeast cells transformed using DNA vectors carrying the HBsAg gene and it is known that the HBsAg synthesized in yeast is assembled in particles with a diameter of 17 to 20 nm and having properties similar to particles of 22 nm secreted by human cells
• the method of the present invention is applicable to yeast cells which are genetically produced and which produce HBsAg.
• step (1) is carried out by the action of mechanical forces such as shear forces (for example the X pressure cell - "X press" - or the French pressure cell) or by stirring with glass beads, possibly in the presence of a detergent.
• mechanical forces such as shear forces (for example the X pressure cell - "X press” - or the French pressure cell) or by stirring with glass beads, possibly in the presence of a detergent.
• Triton X-100 Triton X-100 has been reported by K. MURRAY et al. in The EMBO J. 3; 645-650; 1984 and by R.A. HITZEMANN et al .; loc. cit.
• Steps (2) and (3) include a number of techniques, including adsorption / desorption using colloidal silica, more particularly 1 "'AEROSIL” (a product sold by DEGUSSA, Frankfort, Germany) .
• 'AEROSIL a product sold by DEGUSSA, Frankfort, Germany
• the HBsAg when treating the clarified solution with Aerosil, the HBsAg can be completely desorbed from the solution by using a buffer system of low ionic strength, provided that the additive of the deoxycholate type used so far by an additive of the urea / nonionic detergent type.
• a first objective of the present invention is to provide an improved process for the preparation of hepatitis B surface antigen applicable for vaccine purposes from a culture of genetically produced yeast cells having expressed the 'HBsAg, which process consists to burst the cells in the presence of a nonionic detergent - more particularly a polysorbate (eg 0.3% v / v of polysorbate 20 or 80) and to add urea to it until the final concentration of 2 to 6 M, so as to obtain a clarified solution of HBsAg (eg by stirring at ordinary temperature for 30 minutes), to adsorb the HBsAg on colloidal silica (such as AEROSIL) (eg by stirring at 4 ° C overnight), desorbing HBsAg at 37 ° C at a pH between 8 and 9.5 using a buffer of low ionic strength (eg 10 mM) added urea at a final concentration of 8 M and an ionic detergent, more particularly a polysorbate (eg polysorbate),
• the desorbate contains purified HBsAg particles which can be further processed by any technique well known in the art, eg ultrafiltration, agarose chromatography, precipitation using dextran sulfate / calcium chloride and dialysis.
• a cell pellet (with a dry weight of 80 g), originating from the culture of a yeast strain produced genetically producing HBsAg and which has grown to a production of 30 g of cells (by weight dry) per liter of culture is suspended in 150 ml of an aqueous solution of NazHP0 4 (7.098 g / 1). To this suspension is added 3 ml of a 4% (w / v) EDTA solution, 0.9 ml of polysorbate 20 and 90 ml of isopropanol containing 60 mg of phenylmethylsulfonyl fluoride (FPMS). The pH is adjusted to 8.1 with NaOH (10% w / v in water). The suspension is cooled in an ice bath and broken by two passages through a chilled glass ball homogenizer. The homogenate is then centrifuged for 30 minutes at 13,000 g.
• FPMS phenylmethylsulfonyl fluoride
• the supernatant containing the crude extract (160 ml) is added with urea until a final concentration of 4 M in urea is obtained and stirred for 30 minutes at ordinary temperature so as to separate the HBsAg particles from the cell membranes of contaminating yeast and aEROSIL @ 380 (2.5% w / v) prewashed in 50 mM phosphate buffer (NaH ⁇ PO4 / NaOH), pH 7.2, containing 0.3% (v / v) is added thereto polysorbate 20 and 4 M urea. The solution is stirred at 4 ° C. overnight so as to allow maximum adsorption of the HBsAg particles on the silica particles.
• the colloidal silica is then centrifuged for 15 minutes at 6500 g and washed twice with 250 ml fractions of 10% NaCl (w / v).
• the washed silica is suspended in 80 ml of 10 mM borate buffer, pH 9, (Na 2 B 4 0 7 / HCl), supplemented with 0.5% (v / v) of urea polysorbate 20 (at a final concentration 8 M) and the suspension is stirred at 37 ° for 4 hours.
• the desorbed colloidal silica is then centrifuged for 30 minutes at 27,000 g so as to separate a maximum quantity of colloidal silica particles.
• the desorbate is concentrated up to 15 ml in an AMICON DC device (a device sold by AMICON CORP. DANVERS, MA, EUA) equipped with a cartridge having a cut-off of 100,000 daltons and applied to a column (diameter 2, 5 cm x 100 cm) of SEPHAROSE 4B-CI (an agarose gel manufactured and sold by PHARMACIA FINE CHEMICALS, Uppsala, Sweden) balanced in a 10 mM trometamol / HCl buffer (pH 8) containing 0.3% (v / v) of polysorbate 20.
• AMICON DC device a device sold by AMICON CORP. DANVERS, MA, EUA
• SEPHAROSE 4B-CI an agarose gel manufactured and sold by PHARMACIA FINE CHEMICALS, Uppsala, Sweden
• the excess dextran sulfate is precipitated by dialysis against a solution of 40 mM BaCl 2 in 10 mM trometamol / HCl buffer (pH 8).
• the dialysate (30 ml) is subjected to a centrifugation by gradient on CsCl and the peak containing the HBsAg (9 ml) is collected.
• Electrophoretic analysis shows that the final product is pure at a rate of at least 98% in HBsAg.
• a cell pellet (with a dry weight of 80 g), originating from the culture of a yeast strain produced genetically producing HBsAg and which has grown to a production of 30 g of cells (by weight dry) per liter of culture is suspended in 150 ml of an aqueous solution of Na 2 HP0 4 (7.098 g / I). To this suspension is added 3 ml of a 4% (w / v) EDTA solution, 0.9 ml of polysorbate 20 and 9 ml of isopropanol containing 60 mg of phenylmethylsulfonyl fluoride (FPMS). The pH is adjusted to 8.1 with NaOH (10% w / v in water). The suspension is cooled in an ice bath and broken by 2 passages through a homogenizer with chilled glass beads. The homogenate is then centrifuged for 30 minutes at 13,000 g.
• FPMS phenylmethylsulfonyl fluoride
• the supernatant containing the crude extract (160 ml) is added with urea until a final concentration of 4 M in urea is obtained and stirred for 30 minutes at ordinary temperature so as to separate the HBsAg particles from the cell membranes of contaminating yeast and aEROSIL @ 380 (2.5% w / v) prewashed in 50 mM phosphate buffer (NaHP0 2 / Na 4 0H) pH 7.2, containing 0.3% (v / v) is added thereto ) of polysorbate 20 and 4 M urea. The solution is stirred at 4 ° C. overnight so as to allow maximum adsorption of the HBsAg particles on the silica particles.
• the colloidal silica is then centrifuged for 15 minutes at 6500 g and washed twice with 250 ml fractions of 10% NaCl (w / v).
• the washed silica is suspended in 80 ml of 10 mM borate buffer, pH 9 (Na 2 B 4 0 7 / HCl) supplemented with 0.5% (v / v) of polysorbate 20 and with urea (at a final concentration 8 M) and the suspension is stirred at 37 ° for 4 hours.
• the desorbed colloidal silica is then centrifuged for 30 minutes at 27,000 g so as to separate a maximum quantity of colloidal silica particles.
• the desorbate is dialyzed against a 10 mM trometamol buffer (pH 7.5) in an AMICON DC device fitted with a cartridge having a cut-off of 100,000 daltons in order to dialyze the urea and applied, at a rate of 10 ml per hour, on a 25 ml column of hexylagarose (a product manufactured and sold by PHARMACIA FINE CHEMICALS, Uppsala, Sweden) equilibrated in a 10 mM / HCl trometamol buffer (pH 7.5).
• the column is then washed with 10 mM trometamol buffer (pH 7.5) and eluted with a 50/50 (v / v) mixture of ethylene glycol and 10 mM trometamol / HCl buffer (pH 7.5) added with NaCl at a final concentration of 1 M and then with a 10 mM trometamol / HCl buffer (pH 7.5) added with urea at a final concentration of 4 M.
• Electrophoretic analysis shows that the final product is pure at a rate of at least 97% in HBsAg.
• Example 2 The technique is that of Example 2, but the hexylagarose specified therein is replaced by 25 ml of phenylagarose (a product manufactured by PHARMACIA FINE CHEMICALS, Uppsala, Sweden).
• the HBsAg solution obtained at the end of Example 1 was adjusted to a protein content of 10 ⁇ g per ml by addition of NaCl, phosphate buffer (Na 2 HPO 4 / NaH 2 PO 4 ) and ALHYDROGEL @ (an aluminum hydroxide gel manufactured and sold by SUPERPHOS Export Co., Copenhagen, Denmark) up to respective final concentrations of 0.9% (w / v), 20 mM and 0.15% (w / v ) in AI (OH) s , the final pH being 6.9.
• NaCl phosphate buffer
• ALHYDROGEL @ an aluminum hydroxide gel manufactured and sold by SUPERPHOS Export Co., Copenhagen, Denmark
• the preparation was sterilized and distributed in 2 ml glass vials each containing a unit dose of one ml of vaccine.
• AUSAB @ is the registered trademark for an analysis kit manufactured and sold by ABBOTT Diagnostic Products GmbH, Wiesbaden, FRG.
• the same dilutions of the unit doses of the vaccine preparation, obtained according to the technique of Example 4 but using an antigen prepared according to the technique of Example 1 without the step to colloidal silica were also administered to the 5 groups of five-week-old Swiss mice.

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• 1985
  • 1985-05-30 US US06/739,415 patent/US4649192A/en not_active Expired - Fee Related
• 1986
  • 1986-05-21 EP EP86870069A patent/EP0204680B1/fr not_active Expired
  • 1986-05-21 AT AT86870069T patent/ATE42679T1/de not_active IP Right Cessation
  • 1986-05-21 DE DE8686870069T patent/DE3663108D1/de not_active Expired
  • 1986-05-26 GR GR861357A patent/GR861357B/el unknown
  • 1986-05-26 PT PT82644A patent/PT82644B/pt not_active IP Right Cessation
  • 1986-05-26 AU AU57906/86A patent/AU589331B2/en not_active Ceased
  • 1986-05-26 CA CA000509934A patent/CA1259922A/en not_active Expired
  • 1986-05-27 ZA ZA863935A patent/ZA863935B/xx unknown
  • 1986-05-27 DK DK247586A patent/DK247586A/da not_active Application Discontinuation
  • 1986-05-29 JP JP61125590A patent/JPS61280438A/ja active Granted
  • 1986-05-29 ES ES555442A patent/ES8706444A1/es not_active Expired

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